1. Field of the Invention
The present invention relates generally to the field of diabetes and other glycemic disorders. More particularly, it concerns methods of screening for modulators of glucokinase.
2. Description of Related Art
Non-insulin-dependent diabetes mellitus (NIDDM) is a major public health problem that affects 5–7% of the world population. Although most forms of NIDDM do not exhibit simple Mendelian inheritance, the contribution of heredity to the development of NIDDM has been recognized for many years (Cambridge, 1928; O'Rahilly et al., 1988; Fajans, 1989; Rotter et al., 1990). Early-onset NIDDM or maturity-onset diabetes of the young (MODY) shares many features with the more common form(s) of NIDDM whose onset occurs in midlife (Rotter et al., 1990). In addition, its early age of onset, clear mode of inheritance (autosomal dominant), high penetrance, and the availability of multigenerational pedigrees make MODY an attractive paradigm for genetic studies of NIDDM. Genetic studies have shown tight linkage of early-onset NIDDM and DNA markers on chromosome 20 (Bell et al., 1991) and the glucokinase gene (GCK) on chromosome 7 (Froguel et al., 1992)
At least three distinct mutations have been identified in MODY families (Bell et al., 1996). MODY 2 is characterized by mutations of the glucokinase gene, resulting in a predicted 50–100% decrease in glucokinase activity and impaired insulin secretion (Froguel et al., 1993; Hattersley et al., 1992). The occurrence of diabetes in heterozygous individuals who have some residual glucokinase activity underscores the role of glucokinase as the rate limiting glucose sensor of the beta cell.
Insulin-dependent diabetes mellitus (IDDM), or type 1 diabetes, is a T cell-mediated autoimmune disease. Although, a β-cell lesion is mediated by β-cell specific autoreactive T cells, environmental factors, notably a pancreatotropic virus, may also have an etiological role in this disease. Prevention of diabetes, but not the cure thereof, has been obtained with agents such as insulin, glutamic acid decarboxylase, and hsp60.
The importance of glucokinase (GK) in glucose hemostasis has been demonstrated by the association of GK mutants with diabetes mellitus in humans and by alteration in glucose metabolism in transgenic and gene knockout mice (Froguel et al., 1993; Gidh-Jain et al., 1993; Bali et al., 1995; Hariharan et al., 1997; Postic et al., 1999). Glucokinase (GK) activity is a major determinant of β-cell glucose metabolism and insulin secretion (Sweet et al., 1996; Matschinsky et al., 1998; Matschinsky, 2002). Thus, a complete model of its regulation is central to the understanding of glucose homeostasis and the pathogenesis of diabetic disease states.
To maintain physiological glucose-responsiveness, GK activity needs to be constrained within a very narrow range (Matschinsky et al., 1998; Wang and Iynedjian, 1997). Regulation of GK expression in beta cells has been widely studied and is induced mainly by glucose although it can be modulated by other factors, including insulin (Matschinsky, 2002; Leibiger et al., 2001). Post-translational regulation of GK has only more recently been described, and the mechanisms involved in this mode of regulation are not well known. Recent studies have shown that low levels of glucose cause an association of GK with secretory granules (Toyoda et al., 1999; Stubbs et al., 2000; Rizzo et al., 2002), and that this association correlates with a decrease in GK activity (Rizzo et al., 2002). Prolonged exposure to high glucose (>20 min) causes dissociation of GK from the granule along with conformational changes associated with activation. Glucose-stimulated GK regulation is blocked by inhibitors of insulin secretion, and insulin by itself can rapidly (<2 min) induce similar changes to GK localization and activity (Rizzo et al., 2002). This suggests that minute-to-minute regulation of GK activity and localization occurs through receptor-mediated signaling, and not by interaction between glucose and GK.
The molecular mechanism of GK association with secretory granules and the processes that modulate this association are unknown. The mechanism of GK association with secretory granules have been addressed by examining the role of nitric oxide synthase in its regulation. Neuronal nitric oxide synthase (nNOS) is activated by a rise in intracellular calcium, which is a known response of β cells response to glucose or insulin stimulation (Aspinwall et al., 2000); and nNOS is also known to be localized on insulin secretory granules (Lajoix et al., 2001). Nitric oxide (NO) has also been shown to have a stimulatory affect on glucose stimulated insulin secretion from both cultured β cell lines and pancreatic islets (Smukler et al., 2002; Kaneko et al., 2003). GK is one potential target for regulation by NO, since GK contains several cysteines that have been shown to be critical for maintaining catalytic activity (Tiedge et al., 2000).